On February 27th, to prioritize the development of new and effective antibiotic treatments, the World Health Organization (WHO) released its first Global Priority Pathogens List (global PPL) in 2017; of the 12 pathogens highlighted Streptococcus pneumoniae (Spn) ranked 10th. The global PPL recognizes pathogens that pose a significant threat to global health based on mortality, healthcare and community burden, prevalence of resistance, transmissibility, and treatability. Spn poses a significant threat to global health as this bacterium is no longer susceptible to the major antibiotic penicillin. The main target of antibiotics is the bacterial cell wall, a complex mesh that protects bacteria from an arsenal of both environmental and medical threats. As a Gram-positive ovococcus, Spn, employs two mechanisms to synthesize its protective cell wall: peripheral and septal peptidoglycan (PG) synthesis. These mechanisms require complex, multi-protein machines whose spatiotemporal regulation is largely unknown. Interestingly, Spn lacks the actin homolog and master PG-synthesis-localization orchestrator MreB of rod-shaped bacteria. Curiously, MreB's interaction partner, RodZ, is encoded in the Spn genome and is essential. Note, in rod-shaped bacteria, MreB localization and the subsequent proper localization of the PG synthesis machinery is dependent on RodZ. Since RodZ remains essential in Spn, we speculate RodZ possesses additional functions tangential to binding MreB. Furthermore, in all bacteria, several ambiguities exist regarding the function and localization pattern of RodZ and the position of RodZ in the assembly hierarchy of the PG machinery. The proposed work directly addresses these ambiguities in Spn and thereby will develop new knowledge towards the prevention of disease. Moreover, the proposed work aligns with the NIH's mission to increase global health as cell wall synthesis proteins are promising targets for novel antibiotics. Lastly, greater resolution of the mechanisms by which bacteria construct the cell wall is imperative to global health as antibiotic resistance is on the rise. Hypothesis: We hypothesize that RodZ is a key member of the peripheral PG synthesis machinery and may interact with the actin homolog FtsA to coordinate the localization of the PG synthesis machinery in Spn. The following research aims will elucidate the essential role of RodZ in cell wall synthesis of Spn: Aim 1) Determine key residues essential for RodZ's function in Spn via a reverse genetics approach Aim 2) Characterize RodZ's interacting partners and temporal localization pattern in vivo. For which co- immunoprecipitation experiments and co-localization assays will be used. Aim 3) Investigate RodZ's putative role as a cellular regulator. Towards which end, RNA sequencing and subsequent chromatin or RNA immunoprecipitation sequencing methods will be employed.